Phylogenetic Analysis of Cox I Gene in Identification of Spiders

Journal of Entomology and Zoology Studies 2019; 7(2): 895-897

Jalajakshi S Jalajakshi S and Usha RN Assistant Professor, Genetics Department, Vijaya College, Abstract Basavanagudi, Karnataka, India Morphological diversity refers to diversity of species at the genetic or molecular level. In order to study Usha RN the diversity at the genetic level the taxonomic method DNA barcoding is used. The most commonly Assistant Professor, Biotech used barcode region for animals and some protists is found in mitochondrial DNA (Mt-DNA) i.e. a Department, Mother Teresa portion of the cytochrome oxidase I (Mt-Cox I) gene. The cox gene has a frequency of faster mutation Women’s University, rate and are highly conserved among the species hence Mt-Cox I sequence was used for the practical Kodaikanal, Tamil Nadu, India method of species identification. In the present study, the most dominant female spiders collected were Argiope aemula, Nesticodes rufipes, Oxyopes lineatype, Leucauge decorata, Nephila kuchli, and Nephila philipis. These spiders were preserved in 70% ethanol and DNA was extracted. The amplification of the gene and PCR analysis was done by treating forward and reverse primers. The Cox I gene was sequenced for BLAST sequence similarity search. The phylogenetic analysis revealed the relationship between molecular and morphological taxonomy. The six species with different families have raised from a common ancestor. At each branch point lies the most recent common ancestor of all the groups

descended from that branch point. The four descendents N. rufipes, N. kuchli, N. philipis and O. lineatype

raised from one common ancestor, but O. lineatype emerged as an out group species from the others. Argiope aemula and Laucauge decorata raised from the other common ancestor, indicating the homology sharing.

Keywords: Biodiversity, spider species Mt-Cox I, BLAST sequence, morphological taxonomy, phylogenetic analysis

Introduction

Spiders belonging to the order Araneae of class Arachnida are the most abundant and potential [6] generalist predator of insect pests . Most of them are terrestrial and few are aquatic also. The spiders are different from other insects in, presence of pedipalpi and the head is not differentiated in to different parts (seen in others). The legs of spiders have coxa, trochanter, a patella and a metatarsus. Spiders also have spinnerets and differences in their eyes. The spiders

are the biological agents which capture and eats on many other insects like ants, bugs, mites etc. and thus helps in crop protection. As for as biodiversity of spiders are concerned there is a significant record of the wide variety of species in world, India and also in Karnataka. In the present life science world the word biodiversity is taking a various meanings. Basically it refers to varieties of life forms present on the earth. It is often defined as the totality of genes,

species and ecosystem of a region. Biodiversity is not distributed evenly on earth and is richest in the tropics. Some of the traditional types of biological diversity methods used are taxonomic diversity, ecological diversity, morphological diversity and functional diversity. Morphological diversity refers to the diversity at the genetic or molecular level. In order to study the diversity at the genetic level the taxonomic method DNA barcoding is used. It uses a [4] designated portion of a specific gene or genes to identify an organism to species . The most commonly used barcode region for animals and some protists is found in mitochondrial DNA (Mt-DNA) i.e. a portion of the cytochrome oxidase I (Mt-COX I) gene. The DNA barcoding represents a promising approach to resolve the taxonomic impediment of difficulties in species identification [7]. The Mt-COXI gene sequence is more suitable for the DNA barcoding

because of its faster mutation rate and the sequences are highly conserved among the species. Correspondence The present study aims at, recording the most dominant species from the study area. The Jalajakshi S female spiders were selected to study the morphological diversity, as they exhibit the sexual Assistant Professor, Genetics size dimorphism. The MT-Cox sequence from different families of spiders were used as the Department, Vijaya College, molecular source in order to draw the phylogenetic relationship among the selected species. Basavanagudi, Karnataka, India ~ 895 ~ Journal of Entomology and Zoology Studies

Materials and Methods cTAB was vortexed vigorously and incubated at 60°C for Specimen Collection 1h.To the lysate, 0.5 ml of phenol - chloroform, Iso-amyl Spiders were collected from the surroundings of Turahalli alcohol was added and mixed for 2-3 minutes. It was forest 8km off from the Banashankari temple of South centrifuged at 10000 rpm for 15 min at 4oC. The upper Bangalore. The total area of the forest is 590 acres, with 888 aqueous layer was taken in a new tube, to which double the mt elevation. The coordinates are 12, 88168310 N, volume of cold 100% ethanol was added and 3M sodium 77.52498230 E. The forest is well protected by the Karnataka acetate was added and was incubated for1h minutes at 4⁰C, forest department. The flora and fauna of the area include figs Centrifuged at 10000 rpm for 15 min. The supernatant, was (Ficus tinctoria) neralemara (Syzygium cumini). The most removed and DNA pellet was washed in 70% ethanol and common herb is the Byttneria herbacea. Animals spotted centrifuged at 5000 rpm for 10 minutes. Again the supernatant were Hares Jackals, Lizards, Mongoose, etc. The forests bird was removed, the DNA pellet was air dried and was finally population includes great horned Owls, Mynas, Bablers, and dissolved in TE buffer. more. The variety of spider diversity was found in this region The PCR mixture (final volume of 20 µL) contained 2 µL of hence the area was selected. The collection was done by a DNA, 10 µL of Red Taq Master Mix 2x (Amplicon) and 1µM visual encounter method and hand collection method. Female of each complementary primer specific forward and reverse. spiders were collected, and preserved in 70% ethanol for The samples were denatured at 94oC for 5 minutes, and further usage. amplified using 40 cycles of 94oC for 30 seconds, 44oC for 30 seconds, and 72oC for 1 minute for followed by a final DNA extraction and PCR analysis elongation at 72oC for 10 minutes. The optimal numbers of 100mg of spider tissue was weighed and frozen in dry ice and cycles have been selected for amplification of the gene. the thugh was added with 200 μl of cTAB homogenize 0.5ml

Table 1: Primers used for Cox gene

Gene Primer pair Sequence details Tm Product size (bp) Lco1490FP GGTCAACAAATCATAAAGATATTGG 51.3 Cox 710 hco2198RP TAAACTTCAGGGTGACCAAAAAATCA 53.2

Purification of PCR products unincorporated terminators with an ethanol precipitation Removed unincorporated PCR primers and dNTPs from PCR protocol. The samples were re suspended in distilled water products by using Montage PCR Clean up kit (Millipore). and subjected to electrophoresis in an ABI 3730xl sequencer (Applied Bio systems). Sequencing Phylogenetic analysis: The PCR product was sequenced using the LCO F primers. Based on BLAST analysis the Mt-Cox sequence, of these Sequencing reactions were performed using a ABI PRISM® species has been matched, compared and phylogenetic tree Big Dye TM Terminator Cycle Sequencing Kits with Ampli was constructed. Taq® DNA polymerase (FS enzyme) (Applied Biosystems). The fluorescent-labeled fragments were purified from the Results and Discussion

Fig 1: The Maximum Likelihood method of evolutionary relationship among spiders

In the present study the most dominant female spiders descended from that branch point. The two species Laucauge collected from the study area were, Argiope aemula, decorata and Argiope aemula raised from one common Nesticodes rufipes, Oxyopes lineatypes, Laucauge decorata, branch point. Laucauge decorata belongs to tetragnathidae Nephila kuchli, and Nephila philipis. The Mt- COX I gene family is commonly called as long jawed orb weaver or was sequenced and was used for the practical method of decorative silver orb spider. The body shape and leg shape species identification. The statistical inference, maximum has silver black and yellow marking. Studies have revealed likelihood method revealed the relationship between that evolution of web building has been from irregular to molecular and morphological taxonomy. This method also more regular webs [1]. The web is slanted rather than vertical. involved in finding the evolutionary relationship which yields The Laucauge spider rests in the middle of the web with its the highest probability of evolving the observed data [3]. underside facing upwards. The phylogenetic analysis, was done based on maximum Argiope aemula belongs to Aranidae family exhibits sexual likelihood method. The maximum likelihood method revealed size dimorphism, where females are greatly larger than males. that, the six species with different families have raised from a It shows female gigantism or male dwarfism. Sexual Size common ancestor. The branch point or internal nodes Dimorphism, (SSD) and morphometric analysis of Argiope represent the most recent common ancestor. At each branch anasuja has revealed that the females are four times larger point lies the most recent common ancestor of all the groups than males in their total body length [5]. The web pattern of ~ 896 ~ Journal of Entomology and Zoology Studies these species are in Zig-Zag form resembling the letter, hence 7. Sudhikumar AV, Kashmeera NA. Preliminary Study on they are commonly called as signature spiders. The other four Identification of Spiders Using Mitochondrial DNA. descendants raised from one common ancestor, but O. 2015; 6(8):5741-5743. lineatype belongs to Oxyopidae family is commonly called as jumping spider, golden lynx spiders, emerged as an out group species from the others. The O. lineatype was different from the other species, in not constructing the web and are called as ambush predators. The setae are present on the legs to trap the predators. This could be the reason for O. lineatype to arise as an out group. The branch point represented the most recent common ancestor for orb weavers Nephila kuchli, Nephila philips, and Nesticodes rufipes. N. rufipes emerged out as an out group which belongs to family Therididae and is commonly called as red house spider. It has dark brown globular abdomen, the front half of a spider and legs are red brown in color. Nesticodes builds a small tangled web in dark corners of home or under the rims of garden pots. Extreme sexual size dimorphism species of orb-weaving spiders with large females and small males is seen in Nephila [2]. Nephila kuchli and Nephila Philips belongs to family Nephilidae also has the characteristic feature of exhibiting sexual size dimorphism. Morphologically the legs of Nephila species has alternating dark and light banding pattern. The web pattern is highly complex, builds non sticky, fine meshed barrier webs.

Conclusion The current study demonstrated that phylogeny draws the relationship among individuals or groups of organisms. The biomolecules DNA, RNA and proteins will serve as the sources for the construction of phylogenetic tree. The mitochondrial gene cytochrome oxidase enzyme has a frequency of faster mutation rate and are highly conserved among the species hence can serve as a molecular source for constructing the molecular phylogeny. The bioinformatics software, BLAST programme serves as the search engine for finding the homology between the DNA or proteins from the different organism. Maximum likelihood method marks as an extensive use for the analysis of construction of phylogenetic tree among distantly related sequences.

References 1. Coddington JA. Systematics and evolution of spiders (Araneae). Annu. Rev. Ecol. Syst. 1991; 22:565-592. 2. Elgar MA. Sexual cannibalism, size dimorphism, and courtship behavior in orb-weaving spiders (Araneidae) Evolution. 1991; 45:444-448. 3. Felsenstein J. Evolutionary trees from DNA sequences, a maximum likelihood approach. Journal of molecular Evolution. 1981; 17:368-376. 4. Hebert PDN, Ratnasingham S, deWaard JR. Barcoding animal life: cytochrome c oxidase subunit 1 divergence among closely related species. Proceedings of the Royal Society of London. 2003b; B270:S96-S99. 5. Jalajakshi S, Vinutha, C. Sexual size dimorphism (SSD) and Morphometric analysis of Argiope anasuja (signature spider) of south Bangalore, Karnataka. 2014; 6(04):6059- 6063. 6. Khan AA, Misra DS. Inter and intra specific interaction of wolf spider among predatory arthropods in rice ecosystem. Plant Protection. Bulletin. 2003; 54(3/4):10- 13.

~ 897 ~